A. Absorption and Bioavailability
Absorption refers to the rate and extent by which a pharmacological agent leaves its site of administration and enters the body. Generally speaking, absorption involves the transfer of the pharmacological agent from the site of administration into the blood stream. The route of administration greatly influences the absorption process. For example, because oral administration frequently involves lipid diffusion of the agent across the lining of the gastrointestinal tract, absorption of orally-administered agents is often dependent on the lipid solubility of the agent. By comparison, pharmacological agents administered intravenously bypass the absorption process because they are introduced directly into the vasculature.
The extent to which a pharmacological agent is absorbed frequently does not correlate with the amount of the agent that is able to exert a pharmacological effect. To illustrate, orally administered agents that are absorbed in the gastrointestinal tract pass through the liver prior to reaching the systemic circulation; as a result, those agents that undergo extensive hepatic metabolism (often referred to as "the first-pass effect") will have a much lower effective concentration when they ultimately reach their site of action. In view of the first-pass effect, clinicians are frequently very interested with the parameter of bioavailability, which refers to the extent to which the agent reaches its site of action (or reaches a biological fluid that provides the agent with access to its site of action) see, e.g., Goodman and Gilman's The Pharmacological Basis of Therapeutics (8th edition, L. S. Goodman, A. Gilman, and A. G. Goodman, eds.) Macmillan Publishing Co. Inc., New York, p. 3-18!.
B. Drug Biotransformation
Enzyme systems involved in the biotransformation (metabolism) of a large proportion of xenobiotics (including many commonly administered pharmacological agents) are primarily located in the endoplasmic reticulum of the liver (termed "the microsomal fraction"). In addition, these enzyme systems are located to a lesser extent in, among other places, the epithelium of the Gastrointestinal tract. The biotransformation reactions caused by these enzyme systems result in the first-pass effect mentioned above.
Two major types of enzymatic biotransformation reactions take place. The first type of reactions (phase-I reactions) transform a xenobiotic into a more polar metabolite, frequently through an oxidative process. Oxidative biotransformation reactions are generally conducted by a family of enzymes termed cytochromes P450. The second type of reactions (phase-II or conjugation reactions) couple the xenobiotic (or its polar metabolite) with an endogenous substrate (e.g., glucuronate) see, e.g., Goodman and Gilman's The Pharmacological Basis of Therapeutics (8th edition, L. S. Goodman, A. Gilman, and A. G. Goodman, eds.) Macmillan Publishing Co. Inc., New York, p. 3-18!.
The major cytochromes P450 that participate in the phase-I biotransformation reactions of xenobiotics can be grouped into three families: i) CYP1, ii) CYP2, and iii) CYP3. Enzymes within the CYP3A subfamily are found in hepatocytes and small intestinal epithelial cells (enterocytes) and are extensively involved in phase-I biotransformation reactions.
CYP3A4 is the principal cytochrome P450 present in human liver T. Shimada and F. P. Guengerich, Proc. Natl. Acad. Sci. U.S.A. 86:462-465 (1989)! and enterocytes P. B. Watkins et al., J. Clin. Invest. 80(4):1029-1036 (1987)!. Intestinal CYP3A4 has been implicated in the metabolic elimination of many drugs F. P. Guengerich, Toxicol. Lett. 70:133-138 (1994)!, and first-pass metabolism by the enzyme is believed to contribute to the poor oral bioavailability of some of these drugs see, e.g., J. C. Kolars et al., Lancet 338:1488-1490 (1991); M. F. Paine et al., Clin. Pharmacol. Ther. 60:14-24 (1996)!.
C. In Vitro Models for Studying Oral Bioavailability and Drug Interactions
In vitro studies have previously been performed with the human colon carcinoma cell line Caco-2 see, e.g., M. Pinto et al., Biol. Cell 47:323-330 (1983)! to study oral bioavailability. When grown as monolayers on permeable supports, Caco-2 cells have proven useful as a model for studying intestinal permeability I. J. Hidalgo et al., Gastroenterology 96:736-749 (1989)! and several transport functions, including the transport of bile acids, large neutral amino acids, and some drugs see, e.g., J. Karlsson et al., Br. J. Pharmacol. 110:1009-1016 (1993); D. T. Thwaites et al., Br. J. Pharmacol. 114:981-986 (1995)!.
However, the Caco-2 cell line has thus far fallen short as an ideal model for predicting oral bioavailability or studying drug-drug interactions. For example, cultures of the Caco-2 cell line were capable of metabolizing cyclosporin A to one of the major metabolites, but two other metabolites of cyclosporin A were not detected L.-S. L. Gan et al., Drug Metab. Dispos. 24:344-349 (1996)!. Moreover, the rate of nifedipine oxidation has also been reported to be low in Caco-2 cells X. Boulenc et al., J. Pharmacol. Exp. Ther. 263:1471-1478 (1992)!.
In view of the shortcomings of present models, what is needed is an in vitro model, associated with reproducible results, for accurately predicting bioavailability of pharmacological agents and for studying drug-drug interactions.